During wound healing, fibrin clot is considered as the final step in the coagulation cascade. The process of forming a fibrin clot involves conversion of fibrinogen to fibrin monomers by thrombin (Factor IIa) and cross linking of these fibrin monomers to form a fibrin polymer in the presence of Factor XIII. The resulting fibrin clot acts as a hemostatic plug to seal of the capillaries at the site of injury. During surgical procedures, fibrin sealants are used to aid the surgical closure procedures.
Bergel discovered fibrin's physiological gluing properties in 1909. Since then fibrin has been used as an adhesive. However, it was the clotting property of whole blood that was used and not purified Fibrin. In 1944, Cronkite used fibrinogen component from blood along with thrombin to secure a skin graft but, the low concentrations of fibrinogen and thrombin in the preparation failed to give a good quality clot. In 1975, Maitras was the first to use a concentrated preparation of Fibrinogen for this purpose. Since then the synthetic glues have been increasingly replaced by biological glues. The biological surgical sutures are effective, easy to use and reasonably well tolerated by the patients, but its viral safety, adhesiveness and absence of toxicity to adjacent tissues are the problems still being addressed to varying extents.
The commercial kits consists of components of clot formation like fibrinogen, thrombin, calcium chloride, and an anti-fibrinolytic, typically a plasmin inhibitor. The anti-fibrinolytic (like aprotinin) helps to prevent early degradation of the fibrin clot that may be brought about by plasmin contamination in the fibrinogen-thrombin preparations.
There are several patents in the prior art that disclose processes for the preparation and compositions of concentrates of Fibrinogen and Thrombin in biological glues. Few deal with processes for the preparation of Fibrinogen and Thrombin separately as individual proteins and a few others discuss their processes together as components of a fibrin sealant kit, which mainly involve precipitation of fibrinogen from plasma by the addition of organic solvents or salts at defined concentrations, pH and temperature conditions.
U.S. Pat. No. 5,290,918 and U.S. Pat. No. 5,395,923 disclose processes for the preparation of a protein concentrate coagulable by thrombin, and containing mostly fibrinogen, endogenous Factor XIII and fibronectin. This purification process does not involve any chromatography step.
U.S. Pat. No. 7,550,567 discloses a process for purifying fibrinogen, comprising one or more process steps in which one or more contaminating proteins are depleted by negative chromatography and/or negative adsorption using cation exchanger, hydrophobic gel and/or dye gel. However, absence of plasminogen contamination in the preparation to avoid the use of a protease inhibitor in the Fibrin sealant kit and the final purity levels were not discussed.
WO1997026280A1 discloses a process for the recovery of fibrinogen from a fibrinogen-containing material by affinity. But the plasminogen levels are not quantified in the final preparations.
The second major component in the fibrin sealant kit is Thrombin. Thrombin is obtained by proteolytic cleavage of prothrombin. Purification processes in literature generally describe the purification of prothrombin from plasma and a final single step comprising proteolytic conversion of purified prothrombin to thrombin. Several processes for the purification of prothrombin have been disclosed in prior art. Some of the related patent prior art disclose processes involving protein precipitation alone by addition of salts or other chemicals in one or more steps, while a few other patents disclosed a combination of protein precipitation followed by single or multiple chromatography steps.
U.S. Pat. No. 5,354,682 discloses the purification and recovery of human thrombin produced in commercial-scale quantities. EP 0439156 discloses a process for the production of a liquid thrombin wherein a combination of anion exchange followed by cation exchange chromatography is employed for the purification. U.S. Pat. No. 8,012,728 discloses a process for the preparation of thrombin which is stable in the liquid state. U.S. Pat. No. 5,981,254 discloses a process of preparing biological glue. US 20060134769 discloses a process for the preparation of virus-inactivated thrombin. U.S. Pat. No. 6,245,548 discloses a process for converting pure prothrombin or prothrombin free of other coagulating factors to thrombin by treating prothrombin with sodium citrate. U.S. Pat. No. 5,907,032 discloses a process for the production of thrombin particularly human thrombin that are capable of being produced in a freeze-dried form. The final purity of thrombin and the absence of plasminogen in the preparations have not been elaborated in these patents.
The processes relating to fibrin sealant kits have also been disclosed in prior art, with the kits being made of components like fibrinogen, thrombin, calcium chloride and aprotinin, a protease inhibitor for the preparation of a biological glue. This is exemplified in the inventions described in U.S. Pat. No. 4,427,650, U.S. Pat. No. 5,716,645, U.S. Pat. No. 5,290,918, U.S. Pat. No. 5,395,923, U.S. Pat. No. 5,739,288 and U.S. Pat. No. 5,981,254. Most of the preparations described in the prior art on Fibrin sealant kits are found to be using aprotinin as the anti-fibrinolytic agent to prevent premature lysis of the clot from the contaminating plasminogen in the fibrinogen/thrombin preparations.
Avoiding the use of bovine or synthetic aprotinin can eliminate the risk of hypersensitivity reactions that are known to occur upon repeated exposures to aprotinin as described in a study published in the J Thorac Cardiovasc. Surg. 1998 April; 115(4):883-9). In this, the authors have described a subgroup of patients who developed aprotinin-specific antibodies after topical aprotinin application. The authors suggest that any use of aprotinin in patients should be documented and patients with pre-exposure to aprotinin in any form must be carefully monitored to avoid unexpected anaphylactic reactions. They have even questioned the necessity of adding aprotinin as a stabilizing agent in fibrin sealants. A similar conclusion was drawn by another group that studied the adverse effects of fibrinolysis inhibitor aprotinin in wound healing after suturing tissues with fibrin glue (Biomaterials 24 (2003) 321-327). They showed that even liver tissue which is known to have high fibrinolytic activity was sealed and repaired well in the absence of plasminogen inhibitors. On the contrary, if aprotinin was added, the non-degraded matrix remained in the tissue even after 15 days and affected migration of repair cells. They concluded that the presence of the fibrinolysis inhibitor in the fibrin glue application was detrimental to wound healing.
There are many patents in the prior art—U.S. Pat. No. 7,816,495, U.S. Pat. No. 5,834,420, U.S. Pat. No. 4,022,758, US20130274444, CA 1041424, U.S. Pat. No. 5,792,835, CN 102295696, U.S. Pat. No. 5,138,034, AU 2001023311, DE 19824306, U.S. Pat. No. 6,960,463, U.S. Pat. No. 6,037,457, US 20120195953, AU 199332064, CN 1207064, US 20140154231, U.S. Pat. No. 6,960,463, U.S. Pat. No. 6,960,463, DE19824306, which disclosed purification of fibrin or purification of thrombin but many of them have not disclosed the levels of the contaminants in the final products.